Ecient and facile synthesis of pyrazoles using Guar-gum as biocatalyst and their in vitro bioevaluation

A green ecient and facile synthesis of pyrazoles is reported via condensation reaction between substituted aldehydes (1a-1i), malononitrile (2) and 2,4-dinitrophenyl hydrazine (3) in presence of acylated Guar-gum as biocatalyst under solvent-free conditions. The progress of reaction was checked by thin layer chromatography and melting points reported are uncorrected. All synthesized compounds (4a-4i) were characterized by using 1 HNMR and FTIR spectral techniques and evaluated for in vitro herbicidal activity against Raphanus sativus L. (Radish seeds). All compounds (4a-4i) were also evaluated for their antifungal activity against Rhizoctonia solani and Aspergillus niger by poisoned food techniques method. Antioxidant activity of synthesized compound was also determined. From activity results, it was found that compound 4f was most active against both Raphanus sativus L. (root) and Raphanus sativus L. (shoot) respectively. Compounds 4e and 4h were found most active against Rhizoctonia solani and Aspergillus niger fungus respectively at highest concentration. Compound 4e has shown maximum percentage DPPH free radical scavenging activity 1.e. 61.47% at 100 µg/mL concentration. Less reaction time, excellent yield of products, mild reaction conditions and simple work-up are some merits of present methodology.


Introduction
Recently organic chemists are attracted toward the development of eco-friendly approach for synthesis of biological active heterocyclic functionalities. The development of simple synthetic methods for widely used organic compounds from readily available starting materials is one of the major challenges for organic chemist 1 . Nowadays the use of the biocatalyst for organic synthesis is highly safe that had gained a precious scope in literature. This area of chemistry is a new thought which involves isolation, characterization and synthesis of new compounds and focus mainly on environment safety. Among all the green chemistry aspects selection of green catalyst is an important part of chemical reaction 2 . Nitrogen containing heterocyclic compounds is widely occurring in nature and their applications to biologically active pharmaceuticals, agrochemicals and functional materials are gaining more importance. Thus the development of waste minimized synthetic methods for nitrogen containing heterocycles with structural diversity is one of major eld of interest for organic chemists. Among N-heterocycles pyrazoles is important biologically active heterocyclic compounds and part of natural products. They are mainly used in supramolecular and polymer chemistry, pharmaceuticals, agrochemical industry, food, cosmetic colouring, complexing agents for the synthesis of hydrogenation catalysts and UV stabilizers [3][4][5][6][7][8] . Various methods have been developed for the synthesis of pyrazoles, but most of these methods are not compatible with the environment and also expensive. Therefore, the development of simple and e cient synthesis of pyrazole ring under eco-friendly conditions is highly appreciated [9][10] . In this paper, we reported the synthesis of pyrazoles via one-pot three component reaction between substituted aldehydes, malanonitrile and 2,4-dinitrophenylhydrazine in the presence of Guar-gum. The development of new biocatalysts for synthesis of heterocyclic compounds helps to maximize prevention of waste, high atom economy of products and reduce the use of hazardous chemicals.

Experimental
All chemicals and solvent used were of analytical grade. Melting points were determined on Ganson electric melting point apparatus and are uncorrected. The homogeneity of the reaction was con rmed by (TLC) thin layer chromatography. The 1 HNMR spectra were recorded in CDCl 3 or DMSO-d 6 using tetra methyl silane (TMS) as internal reference on "Brucker Ac 400 F"(400MHz) nuclear magnetic resonance spectrometer. The chemical shifts values were quoted in parts per million. The following abbreviations correlate with the multiplicity of NMR signals: s = singlet, d = doublet, t = triplet, m = multiplet and brs = broad singlet. Infrared spectra (4000-350 cm -1 ) of the synthesized compounds were recorded in KBr pellets on Perkin Elmer FT-IR-R2X spectrophotometer and frequency was expressed in cm -1 .
Acylation of Guar-Gum Take 2 g Guar-gum in 100 mL distilled water in a 250 mL ask and stirred on magnetic stirrer. After the gum was well dispersed, then 10 mL of NaOH solution was added at a rate of 1mL within 15 min with constant stirring at room temperature. An aliquot of 10 mL chloro-acetic acid was then added to the reaction mixture over a period of 10 min. The reaction mixture was heated at 65 ºC with constant stirring for 4 hours to drive the reaction process to completion. The product was separated with ethanol and then added several drops of glacial acetic acid. Finally the precipitate was washed with water and nally freezes and dried 11 .
Chemistry of Acylated Guar-Gum Bioevaluation Screening of herbicidal activity Solutions of 50 µg/ mL, 100 µg/ mL, 150 µg/ mL and 200 µg/ mL of the test compounds in DMSO were prepared. Agar powder (5g) was put into boiling distilled water (1L) until it dissolved, and then cooled down to 40-50 o C. The solution (2 mL) containing test compounds and melting agar (18 mL) was mixed and this mixture was added to a Petridish with 4.5 cm diameter. The agar plate without test compound was used as an untreated control. Then 15 seeds of Raphanus Sativus L. (Radish) were put on the surface of the agar plate. The Petridishes were covered with glass lids, and the cultivation conditions were kept at 25±1 o C and 12 hours in light and 12 hours in dark alternating for seven days. Seven days later, the root lengths and shoot lengths of Raphanus sativus L. were measured. The growth inhibitory rate related to untreated control was determined by given formula 12 .

% inhibition = ((Control -Treated)/Control) x 100
Screening of antifungal activity Amongst the various methods available, poisoned food technique 13 which is the most common was used for testing antifungal activity. The test fungus was grown on Potato dextrose agar medium. The required amount of synthesized compounds dissolved in 1 mL of DMSO was incorporated aseptically into 99 mL aliquots of sterilized potato dextrose agar cooled at 45 o C after brief shaking. Each lot of medium was poured into Petri dishes and allowed to solidify. 1 mL DMSO in media was taken as control. Each dish was inoculated centrally with a 5 mm mycelial disc cut from the periphery of 2-3 days old fungal colonies.
Inoculated Petri plates were incubated in the dark 25±2 o C for 48-72 h and colony diameters were measured periodically till the control dishes were nearly completely covered with fungus growth. Three replicates were used for each concentration of a chemical together with three dishes containing only the solvent and no toxicant. The degree of inhibition of growth was calculated from the mean differences between treatments and the control as percentage of latter by using the formula. Solutions of 25 µg/ mL, 50 µg/ mL, 75 µg/ mL and 100 µg/ mL, of the tested compounds in methanol were prepared. For evaluation of antioxidant activity in 0.2 mL of concentration solution, 3.0 mL of 2,2-diphenyl-1picrylhydrazyl solution was added and mixed thoroughly for 5 min. A control was also made containing 0.2 mL of solvent without concentration of compounds. The absorbance of sample as well as control was measured at 517 nm after 30 min of incubation in dark at room temperature using the UV-visible double beam spectrophotometer model 2203 (Systronics Cop.) against a blank containing respective solvent. A graph was drawn by plotting percent DPPH free radical scavenging activity (y-axis) against given compound concentration (x-axis). Then using the Microsoft Excel Software, a quadratic regression equation (y = ax 2 +bx+c) was obtained. By putting y = 50 in the equation y = ax 2 +bx+c, it was converted to the form ax 2 +bx+c = 0, IC 50 was calculated from the equation above given and using the formula given below: Where X = IC 50 (µg/mL) Calculation of the percentage of DPPH scavenged was calculated by using the formula % DPPH free radical scavenging activity = ((Acontrol -Asample)/Acontrol) x 100 Where, A control = Absorbance of control A sample = Absorbance of sample General method for the synthesis of substituted pyrazoles (4a-4i) A mixture of substituted aldehydes (1a-1i) (20 mmole), malanonitrile (2) (20 mmole) and 2,4-dinitrophenyl hydrazine (3) (20 mmole) was stirred at room temperature in presence of Guar-gum. The progress of the reaction was monitored with the help of thin layer chromatography. After completion of the reaction, the separated solid was ltered and washed with cold water to get the product (4a-4i), which was further recrystallized with ethylacetate and then characterized by 1 H NMR and FTIR spectroscopy.
After completion of the reaction, the solid products were collected by simple ltration and then recrystallized with ethylacetate to afford pure pyrazoles derivatives (4a-4i) as shown in Fig. 1. The physical data of substituted pyrazoles derivatives (4a-4i) was shown in Table 1. The structure of synthesized compounds was con rmed by 1 HNMR, FTIR spectral analysis as well as comparison of their melting points with those of reported compounds. The optimization of reaction conditions has been presented in Table 2. The comparison of activity of different catalysts with respect to time and yield of reaction as shown in Table 3. It was found that Guar-gum gives the best catalytic activity in terms of products yield, reaction conditions and reaction time as compared to other catalyst in literature viz. I 2 /K 2 CO 3 , ZnCl 2 , Al 2 O 3 , NaH, Chloranil and Glycine. The catalyst used in present study is nature derived, easily available and inexpensive which makes this procedure eco-friendly. The plausible mechanism for the formation of substituted pyrazoles derivatives (4a-4i) is shown in Scheme 2. First, nitrile ion is activated by removal of acidic hydrogen from malononitrile catalyzed by acidic catalyst. Finally, the arylidene nitrile intermediate formed through the Knoevenagel condensation reaction between the intermediate nitrile anion and substituted aldehydes. On the other hand, the reaction of 2,4-dinitrophenyl hydrazine with arylidene nitrile afforded an intramolecular cyclized product.     Herbicidal activity All synthesized compounds (4a-4i) were tested for herbicidal activity against Raphanus sativus L. at 200, 150, 100 and 50 µg/mL concentrations (Table 4). Results were shown in the form of primary screening. All compounds were diluted to 1000 µg/mL concentration as a stock solution. Herbicidal activity of compounds was evaluated against Raphanus sativus L. by inhibitory effect of compounds on the growth of weed roots and shoots. The percentage of inhibition growth was calculated from mean differences between treated and control. From the herbicidal activity data, we observed that compound 4f was exhibited maximum percentage growth inhibition i.e. 91.00 against Raphanus sativus L. (root) and also compound 4f was exhibited maximum percentage growth inhibition i.e. 89.07 against Raphanus sativus L.
(shoot) respectively at 200 µg/mL concentration. This growth inhibition may be attributed to substitution of hydroxy group on phenyl ring. The box plot and graphical representation of herbicidal activity of all synthesized compounds (4a-4i) against Raphanus sativus L. were shown in Figs 2-5.

Antifungal activity
All synthesized compounds (4a-4i) were screened for their in vitro antifungal activity against Rhizoctonia solani and Aspergillus niger. The percentage growth inhibition of compounds against R. Solani and A. niger is presented in

Conclusions
A simple and e cient synthesis of substituted pyrazoles (4a-4i) were reported in this paper via condensation reaction between substituted aldehydes (1a-1i), malononitrile (2) and 2,4-dinitrophenyl hydrazine (3) in presence of acylated Guar-gum. Solvent-free medium, non-toxic side products, inexpensive reagents and mild reaction conditions etc. are some beauties of present methodology. All synthesized compounds (4a-4i) were also tested for their bio e cacy in terms of herbicidal activity against Raphanus sativus L. (Radish) seeds, fungicidal activity against R. solani, A. niger and antioxidant activity was also evaluated. Based on activity results, it can be concluded that some of synthesized compounds possessed good activity due to presence of hydroxy, nitro groups on phenyl ring.

Con icts of interest
Authors declare that there is no con ict of interest regarding the publication of this paper.

Funding
Authors received no speci c funding for this study. The funder has no role in study design, data collection and analysis, decision to publish or preparation of the manuscript.